COMET SCGE Assay - Enzo Life Sciences

Data AnalysisWhen excited (425-500 nm) the DNA-bound SYBR ® Green I emits green light. In healthycells the fluorescence is confined to the nucleoid: undamaged DNA is supercoiledand thus does not migrate very far of the nucleoid under the influence of an electriccurrent. In cells that have accrued damage to the DNA, the alkali treatment unwindsthe DNA, releasing fragments that migrate from the cell when subjected to an electricfield. The negatively charged DNA migrates toward the anode and the extrusionlength reflects increasing relaxation of supercoiling, which is indicative of damage.When using TBE as the electrophoresis buffer, the length of the comet tail may becorrelated with DNA damage. When using alkaline electrophoresis conditions, thedistribution of DNA between the tail and the head of the comet should be used toevaluate the degree of DNA damage. The characteristics of the comet tail includinglength, width, and DNA content may also be useful in assessing qualitative differencesin the type of DNA damage.Qualitative AnalysisThe comet tail can be scored according to DNA content (intensity). The control(untreated cells) should be used to determine the characteristics of data for a healthycell. Scoring can then be made according to nominal, medium or high intensity tailDNA content. At least 75 cells should be scored per sample.Quantitative AnalysisThere are several image analysis systems that are suitable for quantitation ofCOMET SCGE Assay data. The more sophisticated systems include the microscope,camera and computer analysis package. These systems can be set up to establish thelength of DNA migration, image length, nuclear size, and calculate the tail moment.At least 75 randomly selected cells should be analyzed per sample.11Assay Designs734.668.6113 or 800.833.8651www.assaydesigns.com